Quorum sensing (QS) is a process of bacterial cell-cell communication that relies on the production, release, detection, and response to extracellular signaling molecules called autoinducers. QS allows groups of bacteria to synchronously alter behavior in response to changes in the population density and species composition of the vicinal community. QS controls collective behaviors including bioluminescence, sporulation, virulence factor production, and biofilm formation.
In pathogenic bacteria that cause persistent infections, QS commonly activates virulence factor production at high cell density (HCD). However, in V. cholerae, which is the etiological agent of the acute disease cholera, production of HapR regulator at HCD represses genes important for virulence factor production and biofilm formation. This peculiar pattern of virulence gene regulation can be understood in terms of the disease. Following successful V. cholerae infection, the ensuing diarrhea washes huge numbers of bacteria from the human intestine into the environment. Thus, expression of genes for virulence and biofilm formation at low cell density (LCD) promotes infection, while repression of these genes by autoinducers at HCD promotes dissemination. Thus, molecules that activate QS have the potential to repress virulence in V. cholerae. March et al reported that pretreatment with commensal E. coli over-producing the V. cholerae autoinducer CAI-1 increased the survival rate of mice following V. cholerae infection [66], which further supports the idea of QS potentiators as drugs.
V. cholerae produces and detects two QS autoinducer molecules called CAI-1 and AI-2. CAI-1 ((S)-3-hydroxytridecan-4-one) is produced by the CqsA synthase and AI-2 ((2S,45)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate) is produced by the LuxS synthase. Detection of CAI-1 and AI-2 occurs through transmembrane receptors CqsS and LuxPQ, respectively. CqsS and LuxPQ are two-component proteins that possess both kinase and phosphatase activities (FIG. 1 shows the CqsA/CqsS system). At LCD, when the receptors are devoid of their respective ligands, their kinase activities predominate, resulting in the phosphorylation of the response regulator LuxO. LuxO˜P is the transcriptional activator of four genes encoding small regulatory RNAs (sRNAs), Qrr1-4. The Qrr sRNAs target the mRNAs encoding the quorum-sensing master transcriptional regulators AphA and HapR. At LCD, facilitated by the RNA chaperone Hfq, Qrr1-4 stabilize and destabilize the aphA and hapR mRNA transcripts, respectively. Therefore, AphA protein is made while HapR protein is not (FIG. 1). When autoinducer concentration increases above the threshold required for detection (which occurs at HCD), binding of the autoinducers to their cognate receptors switches the receptors from kinases to phosphatases (FIG. 1). Phosphate flow through the signal transduction pathway is reversed, resulting in dephosphorylation and inactivation of LuxO. Therefore, at HCD, qrr1-4 are not transcribed, resulting in cessation of translation of aphA and derepression of translation of hapR. This QS circuitry ensures maximal AphA production at LCD and maximal HapR production at HCD. AphA and HapR each control the transcription of hundreds of downstream target genes. Hence, reciprocal gradients of AphA and HapR establish the QS LCD and HCD gene expression programs, respectively (FIG. 1).
Targeting response regulators as a broad-spectrum anti-infective strategy has been considered challenging because response regulator functions, such as phosphorylation and DNA binding, are thought to be specific. In spite of this, a handful of molecules that inhibit particular response regulator functions have been reported. Three inhibitors have been identified that target non-NtrC type response regulators, A1gR1 of Pseudomonas aeruginosa [50], Wa1R in low-GC Gram-positive bacteria [51], and DevR in Mycobacterium tuberculosis [52]. The molecules function by perturbing phosphorylation (A1gR1 and Wa1R) and DNA binding (DevR). Walrycins, molecules that inhibit the phosphorylation of the essential Wa1R response regulator, are active in suppressing growth in multiple Gram-positive bacteria.
LuxO, which is a member of the NtrC family of two-component response regulators, possesses an N-terminal regulatory receiver domain, a central ATPase domain (AAA+type), and a C-terminal DNA-binding domain. Two-component signaling (TCS) proteins are widely distributed in bacteria. In addition to their global importance in microbial physiology, the absence of TCSs in mammalian cells makes them attractive drug targets in pathogenic bacteria. Even though significant effort has been devoted to identifying novel TCS inhibitors, to date, none has been developed into a new class of anti-infective. Problems such as undesirable properties associated with lead molecules have been encountered [56,57]. In particular, inhibitors that generally target the conserved hydrophobic kinase domains of TCS histidine kinases suffer from drawbacks such as low cell permeability, poor selectivity, and unfavorable non-specific off-target effects (e.g. membrane damaging) [58,59,60]. By contrast, approaches to target the sensory domains of histidine kinases have yielded a handful of promising TCS inhibitors. For instance, LED209, an antagonist of the QseC histidine kinase, which regulates motility and pathogenicity in enterohaemorrhagic E. coli, reduces virulence in several pathogens both in vitro and in vivo [61]. In addition, in Staphylococcus aureus, inhibitory Agr peptide analogs antagonize the AgrC histidine kinase receptors and block abscess formation in an experimental murine model [62].